Unusual Membrane-Associated Protein Kinases in Higher Plants

Plant genomes encode a variety of protein kinases, and while some are functional homologues of animal and fungal kinases, others have a novel structure. This review focuses on three groups of unusual membrane-associated plant protein kinases: receptor-like protein kinases (RLKs), calcium-dependent protein kinases (CDPKs), and histidine protein kinases. Animal RLKs have a putative extracellular domain, a single transmembrane domain, and a protein kinase domain. In plants, all of the RLKs identified thus far have serine/threonine signature sequences, rather than the tyrosine-specific signature sequences common to animals. Recent genetic experiments reveal that some of these plant kinases function in development and pathogen resistance. The CDPKs of plants and protozoans are composed of a single polypeptide with a protein kinase domain fused to a C-terminal calmodulin-like domain containing four calcium-binding EF hands. No functional plant homologues of protein kinase C or Ca²⁺/calmodulin-dependent protein kinase have been identified, and no animal or fungal CDPK homologues have been identified. Recently, histidine kinases have been shown to participate in signaling pathways in plants and fungi. ETR1, an Arabidopsis histidine kinase homologue with three transmembrane domains, functions as a receptor for the plant hormone ethylene. G-protein-coupled receptors, which often serve as hormone receptors in animal systems, have not yet been identified in plants. Received: 18 August 1997/Revised: 23 December 1997     

Tetraploids Isatis indigotica are more responsive and adaptable to stresses than the diploid progenitor based on changes in expression patterns of a cold inducible Ii CPK1

In plants, Ca²⁺-dependent protein kinases (CDPKs) are characterized as important sensors of Ca²⁺ flux in response to varieties of biotic and abiotic stress. A comprehensive survey of global gene expression performed by using an Arabidopsis thaliana whole genome Affymetrix gene chip revealed that CDPK tends to be significantly higher in tetraploid Isatis indigotica than in diploid ones. To investigate different CDPK expression in response to polyploidy, a full-length cDNA clone (IiCPK1) encoding CDPK was isolated from the traditional Chinese medicinal herb I. indigotica cDNA library. IiCPK1 contains some basic features of CDPKs: a catalytic kinase domain including an ATP-binding domain and four EFhand calcium-binding motifs. Real-time PCR analysis indicated the expression of IiCPK1 from two kinds of I. indigotica (tetraploid and diploid). They both were induced in response to cold stress, but tetraploids I. indigotica which has good fertility, exhibited an enhanced resistance and higher yield, and presented to be more responsive and adaptable. Our results suggest that IiCPK1 gene plays a role in adapting to the environmental stress.     

Differential accumulation of the transcripts of 22 novel protein kinase genes in Arabidopsis thaliana

22 novel members of the Arabidopsis thaliana protein kinase family (AKs) were identified by using degenerate oligonucleotide primers directed to highly conserved amino acid sequences of the protein kinase (PK) catalytic domain. Of these 22 genes, 16 turned out to carry intron sequences. Homologies of AK sequences were detected to S-locus receptor protein kinases (SRKs) from Brassica spp., to SRK-like PKs from maize and A. thaliana and to several other receptor PKs from A. thaliana. Sequence similarity was also detected to Ca²⁺-dependent PKs (CDPKs) from rape and soybean, to SNF1 and to CDC2 homologues. The genomic organization and the accumulation of the mRNAs from these 22 AK genes were investigated.     

Characterization of Plasmodium falciparum Calcium-dependent Protein Kinase 1 (PfCDPK1) and Its Role in Microneme Secretion during Erythrocyte Invasion

Calcium-dependent protein kinases (CDPKs) play important roles in the life cycle of Plasmodium falciparum and other apicomplexan parasites. CDPKs commonly have an N-terminal kinase domain (KD) and a C-terminal calmodulin-like domain (CamLD) with calcium-binding EF hands. The KD and CamLD are separated by a junction domain (JD). Previous studies on Plasmodium and Toxoplasma CDPKs suggest a role for the JD and CamLD in the regulation of kinase activity. Here, we provide direct evidence for the binding of the CamLD with the P3 region (Leu³⁵⁶ to Thr³⁷⁰) of the JD in the presence of calcium (Ca²⁺). Moreover, site-directed mutagenesis of conserved hydrophobic residues in the JD (F363A/I364A, L356A, and F350A) abrogates functional activity of PfCDPK1, demonstrating the importance of these residues in PfCDPK1 function. Modeling studies suggest that these residues play a role in interaction of the CamLD with the JD. The P3 peptide, which specifically inhibits the functional activity of PfCDPK1, blocks microneme discharge and erythrocyte invasion by P. falciparum merozoites. Purfalcamine, a previously identified specific inhibitor of PfCDPK1, also inhibits microneme discharge and erythrocyte invasion, confirming a role for PfCDPK1 in this process. These studies validate PfCDPK1 as a target for drug development and demonstrate that interfering with its mechanistic regulation may provide a novel approach to design-specific PfCDPK1 inhibitors that limit blood stage parasite growth and clear malaria parasite infections.     

Characterisation and cloning of a calmodulin-like domain protein kinase from Chlamydomonas moewusii (Gerloff )

Calcium-stimulated protein kinase activity in the flagella of the green alga Chlamydomonas moewusii (Gerloff) was characterised. Using SDS-PAGE and an on-blot phosphorylation assay, a 65-kDa protein was identified as the major calcium-stimulated protein kinase. Its activity was directly stimulated by calcium, a characteristic of the calmodulin-like domain protein kinases (CDPKs). Monoclonal antibodies raised against the CDPKα from soybean cross-reacted with the 65-kDa protein in the flagella, and also with other proteins in the flagellum and cell body. The same monoclonal antibodies were used to screen a C. moewusii cDNA expression library in order to isolate CDPK cDNAs from C. moewusii. The CCK1 cDNA encodes a protein with a kinase and calmodulin-like domain linked by a junction domain typical of CDPKs. From Southern analyses, evidence was obtained for a CDPK gene family in C. moewusii and C. reinhardtii. Received: 9 July 1996 / Accepted: 13 November 1996     

Nuclear magnetic resonance structure of calcium-binding protein 1 in a Ca(2+) -bound closed state: Implications for target recognition

Calcium‐binding protein 1 (CaBP1), a neuron‐specific member of the calmodulin (CaM) superfamily, regulates the Ca²⁺‐dependent activity of inositol 1,4,5‐triphosphate receptors (InsP3Rs) and various voltage‐gated Ca²⁺ channels. Here, we present the NMR structure of full‐length CaBP1 with Ca²⁺ bound at the first, third, and fourth EF‐hands. A total of 1250 nuclear Overhauser effect distance measurements and 70 residual dipolar coupling restraints define the overall main chain structure with a root‐mean‐squared deviation of 0.54 Å (N‐domain) and 0.48 Å (C‐domain). The first 18 residues from the N‐terminus in CaBP1 (located upstream of the first EF‐hand) are structurally disordered and solvent exposed. The Ca²⁺‐saturated CaBP1 structure contains two independent domains separated by a flexible central linker similar to that in calmodulin and troponin C. The N‐domain structure of CaBP1 contains two EF‐hands (EF1 and EF2), both in a closed conformation [interhelical angles = 129° (EF1) and 142° (EF2)]. The C‐domain contains EF3 and EF4 in the familiar Ca²⁺‐bound open conformation [interhelical angles = 105° (EF3) and 91° (EF4)]. Surprisingly, the N‐domain adopts the same closed conformation in the presence or absence of Ca²⁺ bound at EF1. The Ca²⁺‐bound closed conformation of EF1 is reminiscent of Ca²⁺‐bound EF‐hands in a closed conformation found in cardiac troponin C and calpain. We propose that the Ca²⁺‐bound closed conformation of EF1 in CaBP1 might undergo an induced‐fit opening only in the presence of a specific target protein, and thus may help explain the highly specialized target binding by CaBP1.     

Purification and characterization of a calcium-dependent protein kinase from beetroot plasma membranes

Several calcium-dependent protein kinases (CDPKs) are located in plant plasma membranes where they phosphorylate enzymes and transporters, like the H⁺-ATPase and water channels, thereby regulating their activities. In order to determine which kinases phosphorylate the H⁺-ATPase, a calcium-dependent kinase was purified from beetroot (Beta vulgaris L.) plasma membranes by anion-exchange chromatography, centrifugation in glycerol gradients and hydrophobic interaction chromatography. The kinetic parameters of this kinase were determined (V _max: 3.5 μmol mg⁻¹ min⁻¹, K _m for ATP: 67 μM, K _m for syntide 2: 15 μM). The kinase showed an optimum pH of 6.8 and a marked dependence on low-micromolar Ca²⁺ concentrations (K _d : 0.77 μM). During the purification procedure, a 63-kDa protein with an isoelectric point of 4.7 was enriched. However, this protein was shown not to be a kinase by mass spectrometry. Kinase activity gels showed that a 50-kDa protein could be responsible for most of the activity in purified kinase preparations. This protein was confirmed to be a CDPK by mass spectrometry, possibly the red beet ortholog of rice CDPK2 and Arabidopsis thaliana CPK9, both found associated with membranes. This kinase was able to phosphorylate purified H⁺-ATPase in a Ca²⁺-dependent manner.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Possible involvement of Ca<Superscript>2+</Superscript>-dependent protein kinases in spore germination of the fern<Emphasis Type="Italic">Osmunda Japonica</Emphasis>

Fern gametophyte is a good model system to investigate signal transduction in plant cells. In this work, we examined whether CDPKs are involved in the mechanisms of spore germination of the fernOsmunda japonica. A protein extract from the spores included four CDPK isoforms with relative molecular weights of 56, 53, 49, and 47 kDa, as detected by immunoblot analysis, and they showed CDPK-like activities, as detected by in-gel protein-kinase assay. It was also found that the inhibitors effective on CDPKs, such as a general protein kinase inhibitor, K252a, and a calmodulin antagonist, W-7, largely suppressed the spore germination, and that many proteins of the spores were phosphorylated in vivo in a calcium dependent manner in the period when the spores require external Ca²⁺ for the germination. Furthermore, we showed that Sr²⁺ and Mn²⁺, which could substitute for Ca²⁺ in the spore germination, were also able to activate theOsmunda CDPKs. From these results, we concluded that CDPKs would participate in the spore germination ofO. japonica.     

Calmodulin isoforms in Arabidopsis encoded by multiple divergent mRNAs

Three new, unique cDNA sequences encoding isoforms of calmodulin (CaM) were isolated from an Arabidopsis cDNA library cloned in gt10. These sequences (ACaM-4, -5, and -6) represent members of the Arabidopsis CaM gene family distinct from the three DNA sequences previously reported. ACaM-4 and -6 encode full-length copies of CaM mRNAs of ca. 0.75 kb. The ACaM-5 sequence encodes a partial length copy of CaM mRNA that is lacking sequences encoding the amino-terminal 10 amino acids of mature CaM and the initiator methionine. The derived amino acid sequence of ACaM-5 is identical to the sequences encoded by two of the previously characterized ACaM cDNAs, and is identical to TCH-1 mRNA, whose accumulation was increased by touch stimulation. The polypeptides encoded by ACaM-4 and -6 differ from that encoded by ACaM-5 by six and two amino acid substititions, respectively. Most of the deduced amino acid sequence substitutions in the Arabidopsis CaM isoforms occurred in the fourth Ca²⁺-binding domain. Polymerase chain reaction amplification assays of ACaM-4, -5 and -6 mRNA sequences indicated that each accumulated in Arabidopsis leaf RNA fractions, but only ACaM-4 and -5 mRNAs were detected in silique total RNA. The six different CaM cDNA sequences each hybridize with unique Eco RI restriction fragments in genomic Southern blots of Arabidopsis DNA, indicating that these sequences were derived from distinct structural genes. Our results suggest that CaM isoforms in Arabidopsis may have evolved to optimize the interaction of this Ca²⁺-receptor protein with specific subsets of response elements.     

Isolation and characterization of a <Emphasis Type="Italic">Paramecium</Emphasis> cDNA clone encoding a putative serine/threonine protein kinase

Mitogen-activated protein (MAP) kinases, a closely related family of protein kinases, are involved in cell cycle regulation and differentiation in yeast and human cells. They have not been documented in ciliates. We used PCR to amplify DNA sequences of a ciliated protozoan—Paramecium caudatum—using primers corresponding to amino acid sequences that are common to MAP kinases. We isolated and sequenced one putative MAP kinase-like serine/threonine kinase cDNA from P. caudatum. This cDNA, called pcstk1 (Paramecium caudatum Serine/Threonine Kinase 1) shared approximately 35% amino acid identity with MAP kinases from yeast. MAP kinases are activated by phosphorylation of specific threonine and tyrosine residues. These two amino acid residues are conserved in the PCSTK1 sequence at positions Thr 159 and Tyr 161. The PSTAIRE motif, which is characteristic of the CDK2 gene family, cannot be found in ORF of PCSTK1. The highest homology score was to human STK9, which contains MAP type kinase domains. Comparisons of expression level have shown that pcstk1 is expressed equally in cells at different stages (sexual and asexual). We discussed the possibility, as in other organisms, that a family of MAP kinase genes exists in P. caudatum.     

Two genes that encode Ca2+-dependent protein kinases are induced by drought and high-salt stresses in Arabidopsis thaliana

Two cDNA clones, AATCDPK1 and cATCDPK2, encoding Ca²⁺-dependent, calmodulin-independent protein kinases (CDPK) were cloned from Arabidopsis thaliana and their nucleotide sequences were determined. Northern blot analysis indicated that the mRNAs corresponding to the ATCDPK1 and ATCDPK2 genes are rapidly induced by drought and high-salt stress but not by low-temperature stress or heat stress. Treatment of Arabidopsis plants with exogenous abscisic acid (ABA) had no effect on the induction of ATCDPK1 or ATCDPK2. These findings suggest that a change in the osmotic potential of the environment can serve as a trigger for the induction of ATCDPK1 and ATCDPK2. Putative proteins encoded by ATCDPK1 and ATCDPK2 which contain open reading frames of 1479 and 1488 bp, respectively, are designated ATCDPK1 and ATCDPK2 and show 52% identity at the amino acid sequence level. ATCDPK1 and ATCDPK2 exhibit significant similarity to a soybean CDPK (51 % and 73%, respectively). Both proteins contain a catalytic domain that is typical of serine/threonine protein kinases and a regulatory domain that is homologous to the Ca²⁺-binding sites of calmodulin. Genomic Southern blot analysis suggests the existence of a few additional genes that are related to ATCDPK1 and ATCDPK2 in the Arabidopsis genome. The ATCDPK2 protein expressed in Escherichia coli was found to phosphorylate casein and myelin basic protein preferentially, relative to a histone substrate, and required Ca²⁺ for activation.     

A calmodulin-stimulated Ca-ATPase from plant vacuolar membranes with a putative regulatory domain at its N-terminus

A cDNA, BCA1, encoding a calmodulin-stimulated Ca²⁺-ATPase in the vacuolar membrane of cauliflower (Brassica oleracea) was isolated based on the sequence of tryptic peptides derived from the purified protein. The BCA1 cDNA shares sequence identity with animal plasma membrane Ca²⁺-ATPases and Arabidopsis thaliana ACA1, that encodes a putative Ca²⁺ pump in the chloroplast envelope. In contrast to the plasma membrane Ca²⁺-ATPases of animal cells, which have a calmodulin-binding domain situated in the carboxy-terminal end of the molecule, the calmodulin-binding domain of BCA1 is situated at the amino terminus of the enzyme.